1. Field of the Invention
This invention refers to production of a synthetic compound termed 4,4′-(4′-Carboxy)-4-nonyloxy-[1,1′-biphenyl]-3,5-diyl)dibutanoic acid (hereinafter referred to as “CNBDA”), and compounds that are derivatives thereof, that inhibit the function of the Src homology phosphotyrosyl phosphatase 2 (SHP2). In addition, the current invention relates to the use of CNBDA as an anti-cancer agent.
2. Description of the Background Art
The Src homology phosphotyrosyl phosphatase 2 (SHP2) is an enzyme that catalyzes dephosphorylation reactions on proteins that bear phosphotyrosine. Structurally, SHP2 has two tandemly-arranged Src homology-2 (SH2) domains in the N-terminal region, a phosphotyrosyl phosphatase (PTP) domain in the C-terminal region and tyrosine phosphorylation sites in the extreme C-terminal tail [1, 2]. The SH2 domains mediate interaction with tyrosine phosphorylated proteins while the PTP domain catalyzes dephosphorylation reactions [3-6]. Specific SH2 domain-mediated interactions and the discriminating capability of the PTP domain confer SHP2 selectivity. The activity of SHP2 is regulated by an intramolecular conformational switch where it assumes a “closed conformation” when inactive and an “open conformation” when active. In the closed conformation, the N-SH2 domain interacts with the PTP domain, physically impeding substrate binding. Engagement of the N-SH2 domain relieves the PTP domain and renders the enzyme active [2]. Mutation of residues that mediate the closed conformation [7] leads to a constitutively active SHP2 found in some human diseases, including Noonan Syndrome and certain leukemia [8-10]. SH2 domain-mediated interaction of SHP2 with Tyr-phosphorylated proteins particularly at the plasma membrane is essential for its phosphatase (PTPase) activity [11-16]. This interaction, not only activates its enzyme function, but also allows its recruitment to target substrates in the cell. Therefore, increased tyrosine phosphorylation in a cell leads to enhanced SHP2 activity. Other events known to activate SHP2 are mutations in the SH2 domains that affect autoinhibition. Some of these mutations result in gain-of-function SHP2 that can promote increased mitogenic and cell survival signaling, leading to human diseases such as Noonan syndrome and hematopoietic malignancies [10, 17, 18]. The disease-causing capacity of activated SHP2 has been experimentally demonstrated where transgenic mice expressing gain-of-function SHP2 mutants develop myeloproliferative disorders [18, 19], supporting the importance of SHP2 in cancer.
In the dephosphorylation reactions of phosphatases, including SHP2, the Asp residue in the so called WPD loop and Cys residue in the active site play essential roles [20]. While the Asp residue acts as a proton donor for the leaving phenolate group of the substrate, the Cys residue executes a nucleophilic attack on the phosphate moiety, leading to hydrolysis of the phosphate moiety. Other residues in the active site of PTPs, also known as the signature motif, such as Arg and Lys residues play substrate binding roles. In addition, Thr and Ser residues in the active site have been shown to play important roles in PTP catalysis [21].
SHP2 is a well-known mediator of mitogenic and cell survival signaling downstream of several signaling pathways [1, 12, 22, 23]. In receptor tyrosine kinase (RTK) signaling, including in epidermal growth factor receptor (EGFR) and human EGFR2 (HER2), SHP2 mediates activation of the Ras-ERK1/2 (extracellular signal regulated kinase 1 and 2) and the PI3K-Akt (phosphatidylinositol 3-kinase—protein kinase B) signaling pathways. One of the mechanisms by which SHP2 mediates Ras activation and consequently of ERK1/2 and PI3K is by blocking the Ras GTPase activating protein (RasGAP), the down regulator of Ras [24, 25]. In EGFR and HER2, SHP2 dephosphorylates phosphorylated Tyr992 (pTyr992) and pTyr1023, respectively, which act as RasGAP binding sites. In Wnt signaling, SHP2 enhances β-catenin signaling by blocking its interaction with α-catenin through dephosphorylation of the latter thereby promoting translocation of the former to the nucleus where it acts as a transcription factor for expression of mitogenic proteins such as c-Myc and cyclin D1 [26]. Similarly, SHP2 promotes Src activation by blocking the translocation of the C-terminal Src kinase (Csk), the Src inhibitor kinase, to the plasma membrane via dephosphorylation of a Csk docking site on PAG (a transmembrane adaptor protein) thereby enhancing Src activation [27]. In all cases, the net effect is increased Tyr kinase signaling to Ras and hence to ERK1/2 and PI3K. It may be this role of SHP2 that allows it to mediate cell transformation induced by oncogenic Tyr kinases [25, 28, 29].
Previous work has shown that SHP2 mediates cell transformation induced by the oncogenic v-Src [29] and the constitutively active form of fibroblast growth factor receptor 3 (K650E-FGFR3) [28]. In addition, SHP2 has been shown to be important for cell transformation induced by the HER2 oncogene [25], and for the growth and transformation phenotype of lung, colon and breast cancer cells [30-32]. These findings suggest that SHP2 plays a cancer promoting role.
The following were the driving forces behind the present invention. First, a large body of literature suggests that SHP2 is a positive mediator of mitogenic and cell survival signaling downstream of the epidermal growth factor receptor (EGFR) [24, 33], HER2, the MET receptor, and several other receptor tyrosine kinase that are dysregulated in breast cancer and many other cancer types [12, 26, 34-38]. Second, recent studies by us and others show that SHP2 is overexpressed in breast cancer with strong association to disease progression and poor clinical outcome [39, 40]. Third, functional studies have shown that SHP2 promotes the transformation phenotype of breast cancer cells and its inhibition suppresses xenograft tumor growth and metastasis [32, 41]. Therefore, inhibition of SHP2 represents a novel therapeutic strategy for the treatment of breast cancer and possibly other cancer characterized by tyrosine kinase activation. To test this possibility, a specific inhibitor of SHP2 must be invented. Based on this real but unmet need, the present invention provides a compound that is an SHP2 inhibitor, derivatives of the compound of this invention, a chemical synthesis scheme for making the compound of this invention, and derivative compounds thereof, and a characterization of the compound of this invention under in vitro and cell culture conditions.